Reactive oxygen species (ROS) have the capacity to cause insulin resistance and oxidative damage. A few specialized cell types have the capacity for extracellular ascorbate cycling. In this process, ascorbate is exported as an antioxidant, and after it is oxidized to dehydroascorbic acid (DHA), the DHA is taken up and reduced intracellularly to ascorbate for another round of antioxidant efflux. We have obtained preliminary data showing that mouse skeletal muscle has the ability to reduce extracellular electron acceptors in a process that is dependent on ascorbate release. Accordingly, the aims of the project are to elucidate the mechanism for, the regulation of, and the physiological consequences of extracellular ascorbate recycling by skeletal muscle. Aim 1 is to determine whether extracellular ascorbate recycling by skeletal muscle provides extracellular antioxidants. The hypotheses for Aim 1 are that muscle cells can reduce extracellular electron acceptors, that the electrons are carried by ascorbate, and that H2O2 plays a countervailing role in this process. Additional hypotheses are that GLUT1-mediated DHA transport supports ascorbate recycling and that anion channels are responsible for extracellular reduction activity and ascorbate efflux. Aim 2 is to determine whether extracellular reduction and ascorbate recycling by skeletal muscle are regulated by insulin and activation of the AMP-activated protein kinase (AMPK). In particular, the aim is to determine whether insulin produces a synergistic increase in DHA transport, glucose 6-phosphate dehydrogenase (G6PD) activity, and ascorbate efflux and to determine whether AMPK stimulates DHA transport but not G6PD activity or ascorbate efflux. Aim 3 is to determine functional roles of ascorbate recycling such as prevention of oxidative damage and maintenance of insulin action. Previously, ascorbate cycling was described for specialized tissues that are small relative to skeletal muscle, which contains 40% of whole-body ascorbate. This project will establish skeletal muscle as a primary generator of extracellular antioxidant and thus an important tissue in whole-body antioxidant status. Further, it could establish the importance of ascorbate cycling and in particular muscle DHA uptake in controlling ROS, thus maintaining normal insulin action. If successful, the project will introduce manipulation of skeletal muscle ascorbate cycling as a novel means for addressing insulin resistance.